Research On Photo-activated Self-degradation Multifunctional DNA Nanoflower Sensitized Tumor Photodynamic Therapy

Photodynamic therapy（PDT）is a safe and non-invasive cancer treatment method.Local laser irradiation activates photosensitizers and generates reactive oxygen species（ROS）to induce tumor cell death.It has shown good application prospects in the treatment of various cancers.However,currently PDT usually requires the use of a higher dose of photosensitizer,which may cause the side effects of photosensitivity.In addition,considering the hypoxia of tumor tissues,low-dose photosensitizers may reduce the oxygen consumption required for PDT,which is essential for alleviating hypoxia-related cell resistance.However,low-dose PDT is less effective in tumor treatment,mainly because the treatment induces obvious cytoprotective autophagy.This is a lysosome-dependent catabolic process that removes organelles and proteins damaged by ROS,and plays a vital role in cell resistance to apoptosis and promoting cell survival.Therefore,there is an urgent need to develop an effective autophagy suppression method to improve the therapeutic effect of low-dose PDT.Based on the above analysis,this project constructed a self-assembled DNA nano-drug（Sgc8c-DNAzyme/DNF-Ce6,SDDC）with photo-activated disintegration,encoding Sgc8c tumor-targeting aptamer,DNAzyme of key autophagy genes,the complement of the Ce6 probe.Specifically,the Sgc8c aptamer is used to target human cervical cancer cells（He La）with over-expressing tyrosine-protein kinase 7（PTK-7）on the cell surface;Ce6 binding sites,and Ce6 labeled DNA probes（Ce6-c DNA）hybridization,by controlling the ratio of DNA nanoflowers and Ce6-c DNA,the loading of Ce6 on DNA nanostructures can be accurately adjusted;DNAzyme is an Mg²⁺-dependent enzyme that can silence autophagy-related gene ATG5 m RNA and significantly damage protective autophagy of tumor cells.On the other hand,the ROS generated by the PDT process can cause DNA strand breaks,triggering the photoactivation and self-degradation of SDDC to release DNAzyme and Mg²⁺.By regulating the loading amount of the photosensitizer Ce6 and the light time,the integrity of the released functional sequence is ensured,so as to realize the autophagy gene silencing effect of amplification in response to light stimulation to sensitize low-dose PDT.The specific research content is as follows:Anti-tumor experiments in vivo and in vitro have shown that SDDC can be specifically taken up in He La cells with high expression of PTK-7 protein,and lysosome escape can be achieved after laser treatment of the cells.At the same time,SDDC can effectively increase the Mg²⁺level in He La cells and provide a sufficient number of auxiliary ions to activate DNAzyme.SDDC can effectively produce ROS in He La cells to achieve cell killing effect,but it was less toxic to normal cells Hs578Bst.RT-q PCR and Western blot experiments showed that photoactivated autolytic SDDC can silence the expression of autophagy genes and proteins in He La cells.After injection through the tail vein,SDDC was specifically taken up by tumor cells through PTK-7-mediated endocytosis.After 14 days of administration the next day,the tumor growth of the mice was significantly inhibited.No significant weight loss was observed in mice in each experimental group,indicating that the systemic toxicity of low-dose PDT was negligible.This subject systematically studied the photodynamic therapy of amplified gene silencing to enhance tumor sensitization by light-activated self-degrading multifunctional DNA nanoflowers.In vivo and in vitro experiments have shown that SDDC can effectively target tumor tissues,using low-dose photosensitizers to achieve significant anti-tumor therapeutic effects.This rationally designed photo-activated self-degradation strategy provides a new way to overcome the low bioavailability of oligonucleotide drugs delivered by DNA nanostructures,and at the same time demonstrates the great potential of inhibiting the beneficial autophagy of tumor cells to sensitize low-dose PDT.